Polymerase chain reaction (“PCR”) is a technique widely used in molecular biology. It derives its name from one of its key components, a DNA polymerase used to amplify a piece of DNA by enzymatic replication. Typically, PCR employs a thermostable polymerase, deoxynucleotide triphosphates (“dNTPs”), a pair of primers, and a template DNA. A single PCR reaction (or cycle) often involves (1) increasing the sample temperature to a temperature sufficient to melt or denature a double-stranded DNA molecule into single-stranded templates, (2) cooling the sample to allow a DNA primer to bind or anneal to each template, and optionally (3) re-adjusting the sample temperature to optimize the enzymatic addition of dNTPs onto a terminus of each bound primer to form a new DNA molecule. As PCR progresses, the generated DNA (the “amplicon”) is itself used as a template for further replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. With PCR, it is possible to amplify a single or few copies of a DNA across multiple orders of magnitude, generating millions or more copies of the DNA.
Efficient PCR depends on accurately and reproducibly reaching product/template denaturation (or melting) and primer annealing temperatures during thermal cycling. This, in turn, depends on accurately measuring and controlling the sample and/or solution temperature. PCR sample temperature measurement and control can be performed manually or through automated instrumentation such as a thermal cycler (or thermocycler). Temperature sensors in many thermal cycling instruments measure the temperature of the metal block or air chamber surrounding the PCR tube that contains the amplification solution. With such “external” temperature sensors, accurate measurements can sometimes be obtained during equilibrium when the temperature is held constant. During temperature transitions, however, the solution temperature frequently lags behind the instrument block or chamber temperature, potentially leading to inaccuracy and inconsistency in temperature monitoring and control—an effect that becomes even more pronounced as the PCR cycling speeds increase.
Direct sensor contact within the PCR solution, while potentially more accurate than external temperature measurement, also can be problematic. Such direct, internal measurement of PCR is often disfavored because of product contamination, PCR inhibition, added thermal mass of the sensor, and obstruction of optical measurements. Many of these concerns become more acute as the sample volume decreases. In larger samples, however, direct physical sensors measure the temperature of only one location that may not accurately reflect the temperature of the entire solution.
Over time, temperature cycling for PCR has become faster. At faster speeds, most of the cycle time is spent in temperature transition, and the solution temperature seldom tracks the measured instrument temperature. Attempts to improve identifying the solution temperature during fast PCR cycling include prediction algorithms that depend on sample volume and sensors with the same thermal response as the samples so that they are kinetically matched. However, since the biochemical reactions in PCR are rapid and there are many ways to change the temperature of a sample quickly (especially small samples), the limiting factor for consistent PCR (especially at fast speeds) appears to be accurate temperature measurement.